Simultaneous Assay for Multiple Pathogens in Blood (SAMP-B)

摘要 快速、高通量和高度准确地识别潜在的血液传播疾病将有助于血库快速检测非常大量的捐献血液，并有助于防止进一步感染。目前的血液检测存在以下问题： 1.所有疾病都必须逐一进行耗时的检测和样本操作。 2.根据测试次数的不同，需要大量的样本。 3.测试次数越多，测试程序越复杂。 4.复杂的操作程序，必须由训练有素的分子实验室人员操作。 最近使用核酸扩增检测技术(NAT)的商业化检测技术的发展使血液中心考虑将这些检测应用于献血者筛查。但到目前为止，NAT测试系统是时间密集型的，不是“多路传输(&gt；2)方式”，因此不适合大规模筛查捐赠者样本中的各种病原体。 Maxwell Sensors，Inc.(MSI)建议将解旋酶依赖的放大和条形码磁珠(BMB)技术相结合，以产生对血液中多种病原体的快速、同时检测(SAMP-B)。只需在单个微孔中滴几滴捐献者的血液和数字磁珠，就可以同时识别艾滋病毒1型和2型、乙肝(乙肝)、梅毒、丙型肝炎(丙型)、人类T淋巴细胞病毒(HTLV I和II型)、西尼罗河病毒(WNV)和许多其他血液传播疾病。在这一阶段的项目中，我们完成了所有的任务，并成功地展示了多个病原体同时检测的技术可行性，制作了128个复合BMB，集成了分析仪，并进行了艾滋病毒、乙肝和梅毒的多重检测。由此产生的组合技术非常强大，并将提供以下优势： (1)样本数量少：只需在微孔中滴几滴血就可以识别多个目标病原体。它 不仅可以确定反应性样本，还可以确定“对什么反应” (2)缩短窗口期：提供高灵敏度和高特异度，而无需与基于抗体的筛查技术相关的“窗口期” (3)快速、高吞吐量：该系统每小时可自动处理多达100个样本-最多12,800个96微孔格式的可报告结果 (4)灵活性：易于添加新的探头，以增加珠子上的筛查目标 (5)成本低、使用方便、可靠性高：BMB成本低，操作步骤简单，批次差异小。 在这一二期项目中，我们将专注于：(1)将血液传播病原体鉴定板从3-plex扩大到8-plex，(2)将样本流程与SAMP-B分析仪集成，以及(3)开发和评估用于血液筛查的多重病原体。

ABSTRACT Rapid, high-throughput, and highly accurate identification of potential blood borne diseases will help blood banks to quickly test a very large volume of donated blood, and will help to prevent further infections. Current blood testing has the following issues: 1. Time consuming testing and sample operation that must be performed for all diseases "one-by-one". 2. A large sample volume is required that depends on the number of tests. 3. Testing procedure is complicated as the number of tests increase. 4. Complicated operational procedures that must be run by highly trained personnel in molecular laboratories. The recent development of commercially-available assays using nucleic acid amplification test technologies (NAT) have made it possible for blood centers to consider applying these tests to blood donor screening. But NAT test systems, so far, are time intensive, not in "multiplexed (> 2) fashion", thus are unsuited to large-scale screening of donor samples for various pathogens. Maxwell Sensors, Inc. (MSI) proposes to combine Helicase-Dependent Amplification and Barcode Magnetic Bead (BMB) technologies to produce a rapid, Simultaneous Assay for Multiple Pathogens in Blood (SAMP-B). With a few drops of a donor's blood combined with digital magnetic beads in a single microwell, it is possible to simultaneously identify HIV type 1 and 2, Hepatitis B (HBV), Syphilis, Hepatitis C (HCV), Human T-Lymphotropic Virus (HTLV type I and II), West Nile Virus (WNV), and many other blood-borne diseases. During this Phase I project, we have completed all tasks and successfully demonstrated the technical feasibility of simultaneous assay for multiple pathogens by fabricating 128-plex BMBs, integrating the analyzer, and performing HIV, HBV, and Syphilis multiplexed assays. The resulting combined technology is very powerful and will offer the following advantages: (1) Small quantity of sample: a few drops of blood in a microwell are all that's needed to identify multiple target pathogens. It not only determines a reactive sample, but also identifies "reactive to what" (2) Reduced window period: offers high sensitivity and specificity without the "window period" associated with antibody based screening technologies (3) Rapid and high throughput: the system can automatically process up to 100 samples per hour - for a maximum of 12,800 reportable results in a 96-microwell format (4) Flexibility: easy addition of new probes for additional screening targets on beads (5) Low cost, easy to use, and high reliability: BMB are low cost, simple operation steps, and batch to batch variation is minimal. In this Phase II project, we will focus on: (1) expand blood-borne pathogens panels from (3-plex to 8-plex) for Blood screening, (2) integrate the sample process with SAMP-B analyzer, and (3) develop and evaluate multiplex pathogens for blood screening.